ESTRO 36 Abstract Book

the 3D reconstructs to study radiation effects on the immune system. PO-0958 Radiogenomics: role of non-coding RNA genes in increased radiotherapy sensitivity L. Duran-Lozano 1 , V. Reyes 2 , M. Mollà 2 , M.J. Fuentes- Raspall 3 , M. Altabas 2 , T. Ramón y Cajal 4 , A. Barnadas 4 , O. Diez 1,5 , J. Giralt 2 , S. Gutiérrez-Enríquez 1 1 Vall d'Hebron Institute of Oncology-VHIO, Oncogenetics Group, Barcelona, Spain 2 Vall d'Hebron University Hospital, Department of Radiation Oncology, Barcelona, Spain 3 Hospital de la Santa Creu i Sant Pau, Department of Radiation Oncology, Barcelona, Spain 4 Hospital de la Santa Creu i Sant Pau, Medical Oncology Department, Barcelona, Spain 5 Vall d'Hebron University Hospital, Area of Clinical and Molecular Genetics, Barcelona, Spain Purpose or Objective Breast cancer (BC) is the first cause of cancer-related mortality of Spanish women and most common cancer in women worldwide. It is frequently treated with radiotherapy (RT), which can cause early and late side- effects that impact negatively on quality-of-life of cancer survivors. MicroRNAs and long non-coding RNAs (lncRNAs) modulate key cellular pathways in response to radiation. Single nucleotide polymorphisms (SNPs) in these two types of non-coding RNAs can alter their function and consequently modify the expression of genes that regulate, affecting the respective biological activities. As part of a long-term ongoing study, our aims were to test genetic association of SNPs in microRNAs and lncRNAs with late radiotherapy-induced toxicity and to characterize the expression of lncRNAs in blood cells of BC treated patients. Our final goal is to discover new genetic endpoints to predict individuals with increased susceptibility to radiotherapy side effects. Material and Methods DNA samples and clinical data were collected from 198 prospectively and 72 retrospectively recruited BC patients treated with RT in two hospitals. All patients were followed at least between two and six years after RT. 34 SNPs in microRNAs and lncRNA genes related to radiation response were genotyped using iPLEX® Gold with MassArray Agena Bioscience (Sequenom). RNA was obtained from blood before and after radiotherapy of 19 BC patients from the prospective cohort. Eight lncRNAs (FAS-AS1, MALAT1, TP53TG1, HOTAIR, PANDA, MEG3, ANRIL and LINC00467) involved in radiation cell response were assessed by RT-PCR, agarose gels and direct sequencing. A semiquantitative capillary electrophoresis of fluorescent amplicons was performed to estimate the proportion of total transcripts. Results The first analysis showed an association of overall long term toxicity after radiotherapy with rs4559081 A/A genotype of LINC00336 (OR=3.91 95%CI = 1.34-11.37), and grade ≥ 2 late radiation skin toxicity (fibrosis or telangiectasias) with rs17762938 A/C or CC of PCAT1 (OR=2.63 95% CI=1.00-6.86) and rs2910164 C/G of miR- 146a (OR=0.27 95%CI = 0.008-0.94). The expression and presence of different isoforms of all lncRNAs evaluated, except ANRIL and HOTAIR, were observed in blood cells. After radiotherapy the level of MEG3 and PANDA expression increased. Conclusion The potential genetic association of the lncRNAs LINC00336 and PCAT1, and microRNA miR-146a with radiotherapy-induced late toxicity needs to be confirmed in larger breast cancer cohorts. The expression of lncRNAs can be a biomarker of radiotherapy response measurable in blood. Funding: This research was supported by a grant [FIS 05/2181] from ‘‘Fondo de Investigación Sanitaria,

Instituto de Salud Carlos III, Ministerio Español de Economía y Competitividad”. PO-0959 REQUITE: Radiation Induced Lymphocyte Apoptosis assay as a predictor for radiotherapy side effects C. Talbot 1 , A. Appanvel 2 , A. Botma 2 , T. Rancati 3 , A. Webb 1 , D. Azria 4 , T. Burr 5 , J. Chang-Claude 2 , C. Herskind 6 , D. De Ruysscher 7 , R. Elliott 8 , S. Gutiérrez Enríquez 9 , P. Lambin 7 , B. Rosenstein 10 , T. Rattay 11 , A. Vega 12 , F. Wenz 6 , R. Valdagni 3 , C. West 8 1 University of Leicester, Department of Genetics, Leicester, United Kingdom 2 German Cancer Research Centre DKFZ, Genetic Epidemiology Unit, Heidelberg, Germany 3 Fondazione IRCCS Istituto Nazionale dei Tumori, Prostate Cancer Program, Milan, Italy 4 University of Montpellier, Institut du cancer de Montpellier, Montpellier, France 5 Source Bioscience, R&D, Nottingham, United Kingdom 6 Heidelberg University, Department of Radiation Oncology, Mannheim, Germany 7 Maastricht University Medical Center, Department of Radiation Oncology MAASTRO clinic, Maastricht, The Netherlands 8 University of Manchester, Institute of Cancer Sciences, Manchester, United Kingdom 9 Vall d’Hebron Institute of Oncology-VHIO, Radiation Oncology Department, Barcelona, Spain 10 Mount Sinai School of Medicine, Department of Radiation Oncology, New York, USA 11 University of Leicester, Department of Cancer Studies, Leicester, United Kingdom 12 Universidade de Santiago de Compostela, Centro de Investigación Biomédica en Red de Enfermedades Raras CIBERER, Santiago de Compostela, Spain Purpose or Objective Recently the first replicated genetic associations for radiotherapy-induced adverse reactions were reported. The European Union funded REQUITE consortium aims to validate known predictors of adverse reactions to develop clinically useful tools. One such predictor is low levels of radiation induced lymphocyte apoptosis which has previously been found in patients experiencing increased rates of late radiation induced toxicity. Material and Methods REQUITE is a multi-centre, observational study. Enrolment was open for two and a half years in nine centres (eight in Europe and one in the United States), with another two years of follow-up still ongoing. The primary endpoints are change in breast appearance at two years (breast), rectal bleeding at two years (prostate) and breathlessness at 12 months (lung). Work Package 4 involves validation of biomarkers. This includes genetic polymorphisms and the radiation induced lymphocyte apoptosis assay (RILA). The RILA was carried out in three of the European centres using a standardised protocol which had been verified with inter lab testing; it assesses percentage radiation induced apoptosis in lymphocytes, detected by flow cytometry, 48 hours after ex-vivo irradiation of whole blood. Results More than 4300 patients have been enrolled in REQUITE. 1322 samples have been analysed using the apoptosis assay. The levels of apoptosis 48 hours after ex-vivo irradiation increase over baseline in a range from 2.4% to 62.4%, confirming large inter-patient variability. In the Leicester cohort mean RILA is higher in the prostate patients compared to the breast patients (24.9% vs 20.3%; p=0.004). Analysis of predictive value for acute toxicity is being carried out. Conclusion Variation in percentage of lymphocyte apoptosis is in keeping with previous studies. This large scale prospective observational study will be the largest to date to assess